Investigations of the localisation of bacterial activity on sandstone from ancient monuments

Methods were investigated for the determination of activity levels of bacteria on sandstone using the reduction of 2-(4-iodophenyl)-3-(4 nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan as a measure of dehydrogenase activity. A microscopy technique, based on use of acridine orange with bright-field illumination, was developed which gave a good visual image of bacterial cells, including those containing INT-formazan. Surveys at two monuments, Portchester Castle and Tintern Abbey, were carried out using this method which showed that between 20.7 and 51.9% of bacterial cells present in situ were active. Extraction of INT-formazan directly from the stone using methanol indicated that bacteria were tightly bound to stone particles and that microscopic methods would underestimate counts of bacteria. Surveys of five monuments using the extraction method showed that microbial populations on sandstone in situ were active but activity could not be related to decay state of the stone.